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Abstract

Cell wall is a complex biopolymer on the surface of all Gram-positive bacteria. During infection, cell wall is recognized by the innate immune receptor Toll-like receptor 2 causing intense inflammation and tissue damage. In animal models, cell wall traffics from the blood stream to many organs in the body, including brain, heart, placenta and fetus. This protocol describes how to prepare purified cell wall from Streptococcus pneumoniae, detect its distribution in animal tissues, and study the tissue response using the placenta and fetal brain as examples.

Host response to infection involves recognition of many bacterial components including the cell wall (CW), a complex macromolecule that forms the surface of all Gram-positive bacteria. The CW of Gram-positive bacteria is formed by the covalent network of peptidoglycan and teichoic acid. Streptococcus pneumoniae, a leading cause of pneumonia, sepsis, and meningitis, has served as an important model organism for studying the innate immune response to Gram-positive bacterial infection including CW.

This protocol describes how to prepare purified CW from Streptococcus pneumoniae (Tuomanen et al., 1985b) and follow its distribution in mice after intravenous injection, focusing on the placenta and fetal brain as examples (Humann et al., 2016). This model yields histopathologic sections of organs for study of the tissue response to CW components. Our model’s focus on pneumococcal CW derives from its well-described role in inflammation and injury in many organs, its extensive known chemistry and its recognition as a classic TLR2 pathogen associated molecular pattern.

Prepare all the solutions and do all washes with distilled water (use cell culture distilled water from Sigma [#W3500] with low endotoxin concentration).

Filter all the solutions before use (with filter 0.22 µm).

Preferably use new clean glassware and equipment to avoid any contamination with endotoxin (LPS).

Prior to large-scale growth, check pneumococcal stock on blood plate without antibiotics to confirm the absence of contamination.

The protocol described here uses C+Y medium (see Recipes) as a partially defined growth medium. Other commercially available media are also suitable but have more diverse contaminating components such as endotoxin that needs to be removed for achieving a pure preparation.

Preparation of stock of Streptococcus pneumoniae
CW can be purified from any pneumococcal strain. However, the procedure is most successful with unencapsulated strains such as R6 (Tuomanen et al., 1985b).

Figure 1. Overnight growth of unencapsulated strain R6 on TSA blood agar. Pure culture of pneumococcus is the source of bacteria for seed stocks made in step A1b.

Scrape all the colonies off the agar plate with an inoculation loop, and inoculate into 10 ml C+Y medium in a test tube. Incubate the culture at 37 °C, 5% CO2. When the optical density of the culture at 620 nm (OD620) reaches 0.4, harvest the culture by centrifugation at 1,500 x g, 4 °C for 10 min in a microcentrifuge.

To estimate bacterial growth in flasks, transfer one 10 ml aliquot (using sterile serological pipet) from flask into a sterile glass tube when the culture begins to appear turbid. Read the culture turbidity at 620 nm (OD620) of this transferred culture. Keep the bacterial culture in the glass tube, and incubate it in the incubator with the flask culture. Monitor the OD620 of the glass tube culture over time.

OD620 in flask will be approximately 0.1 higher than OD620 in glass tube.

When the OD620 in the flask reaches 0.7-0.8, remove the flask from the incubator and immerse it into ice for 15 min. Swirl the flask every 2 min to cool culture rapidly.

Pour the cooled culture into pre-chilled 1,000 ml centrifuge bottles, and harvest the culture by centrifugation at 4 °C, 4,000 x g for 10 min in the Sorvall centrifuge.

Decant supernatant carefully by pouring. Pellets can be stored at -80 °C until use

Preliminary harvest and mechanical breakage of the bacterial cellsNote: The operational definition of crude CW is material that is precipitable by boiling in SDS.

Resuspend the pellet in 20 ml/tube 1 M NaCl (see Recipes). Centrifuge the suspension at room temperature, 12,000 x g for 10 min in the Sorvall centrifuge. Discard the supernatant gently and carefully. Repeat this step for another 2 times.

Resuspend the pellet in 20 ml/tube water. Centrifuge the suspension at room temperature, 12,000 x g for 10 min in the Sorvall centrifuge. Discard the supernatant gently and carefully. Repeat this step for another 7 times to remove detergent.

Add 10 ml water to the pellet and vortex vigorously. Incubate the suspension at room temperature for 20 min to separate supernatant by sedimentation. Then, remove the supernatant carefully with a sterile serological pipette. Transfer the supernatant into the centrifuge tube in step A4b. Repeat this step at least 5 times until the supernatant appears clear.

Resuspend the pellet in 10 ml water, centrifuge the suspension at room temperature, 27,000 x g for 15 min in the Sorvall centrifuge. Discard the supernatant.

Resuspend the pellet in 10 ml acetone, centrifuge the suspension at room temperature, 27,000 x g for 15 min in the Sorvall centrifuge. Discard the supernatant very carefully because the pellet is very loose.

Resuspend the pellet in 10 ml water, centrifuge the suspension at room temperature, 27,000 x g for 15 min in the Sorvall centrifuge. Discard the supernatant. Repeat this step for another 5 times.

Resuspend the pellet in 2 ml water.

Test the CW suspension for endotoxin by PierceTM LAL Chromogenic Endotoxin Quantitation Kit according to the manufacturer’s instruction.

Record the weight of the microcentrifuge tubes, and aliquot the CW suspension into the tubes.

Lyophilize the CW suspension in a Speed-vac.

Record the weight of the tube containing lyophilized CW. Subtract the weight of the empty tube to obtain mass of CW. Reconstitute the dried CW by adding 348 μl H2O per 46 mg CW material, which results in CW stock equivalent to 106 cfu/µl. Store the CW stocks at 4 °C.

The composition of this CW material has been described (Holtje et al., 1975).Notes: The CW stock will have a ‘milky’ appearance (Figure 2).

Figure 2. Final appearance of purified pneumococcal CW

FITC labeling of CW

Pipet the desired amount (100-200 μl depending on the number of mice) of CW from the CW stock into a fresh microcentrifuge tube.

Centrifuge the FITC-labeled CW at room temperature, 17,000 x g for 2 min in a microcentrifuge. Discard the supernatant, and resuspend in 1 ml DPBS. Repeat this step 2 times.

Resuspend the FITC-labeled CW pellet in DPBS to a concentration of 1 x 106 bacterial equivalents/μl.

Figure 3. Final appearance of FITC labeled pneumococcal CW

Detection of CW distribution in placenta and fetal tissues
All experiments should be performed in compliance with national and institutional guidelines (for example, National Institute of Health).

General description
To study CW distribution to the placenta and fetus in mice, FITC labeled CW is injected intravenously into gestating mice. Male and female mice are purchased and bred in the animal facility on-site. Healthy mice at a body weight of 15-20 g and no signs of dehydration or illness are anesthetized by isofluorane. The mice are kept under isofluorane throughout the duration of the procedure (~5-10 min). Mice recover from the 10 min procedure in ~15-20 min.

In the surgical suite, the mouse is warmed on a heating pad 10-15 min before injection to dilate blood vessels. The mouse is then restrained with tail access.

Bacterial equivalents of 2 x 107 of FITC CW (20 μl of the FITC CW preparation) is diluted into a total volume of 100 μl sterile PBS and injected into the tail vein. The injection is done using a 25 gauge needle.

For recovery, the mice are moved to a recovery table and monitored by respiration rate, toe pinch for capillary refill time and a heating pad for maintaining body temp. They are monitored visually for at least 30 min post-operationally until they are moving freely in the cage to assure they can access water and food.

Harvesting tissues at Embryonic Day E18-20

Mice are euthanized and placenta and embryo heads are excised and fixed overnight at 4 °C in 4% paraformaldehyde (PFA) in a 50 ml conical tube.

Drain the PFA and fill the tube with 30% sucrose. Tissue should initially float at the top of the tube.

Keep tissue in sucrose at 4 °C for 3-5 days until tissue rests at or near the bottom of the tube.

Remove tissue one at a time to a clean 10 cm dish. Cut with a razor blade according to the diagram in Figure 4 for either coronal or sagittal sectioning.

Visualization of CW in the placenta and fetal tissues

Figure 4. Cross sections of mouse brain. For sectioning, make incision along dotted line. Coronal section is preferred to remove cerebellum and create flat surface for embedding the cortex.

Place in embedding mold with cut surface on bottom of mold. Add OCT compound slowly to cover, avoiding bubbles.

Once dried, apply a thin layer of Prolong Gold anti-fade mounting medium with DAPI and carefully cover with a glass coverslip to avoid introducing air bubbles.

Allow slides to cure overnight at room temperature and analyze the following day with confocal microscope.
Fluorescent images were acquired with an inverted confocal microscope (LSM510; Carl Zeiss MicroImaging, Inc.). Argon laser (excitation 488 nm) and a filter set to detect FITC emission (BP500 – 550) for imaging FITC; Chameleon laser (excitation 740 nm) and a filter set to detect DAPI emission (BP435 – 485) for imaging DAPI. Cells were observed using a 20x/0.75 Plan Neofluor objective with the confocal pinhole set to one Airy unit. Imaging parameters, such as gain and offset levels, and line averaging, are optimized to avoid oversaturation of pixels and to improve signal:noise ratio. Images were acquired using Zen 2008 software package (Carl Zeiss MicroImaging, Inc.). CW pieces per section were counted using the particle analysis tool in ImageJ (imagej.net/Particle_Analysis)

Data analysis

Figure 5. FITC CW in the fetal brain or placenta is documented and quantified by microscopy. Confocal images of CW distribution in placental and fetal brain tissue (arrows) are analyzed using ImageJ.

Three independent experiments are carried out for each treatment condition and the results are combined. Data are analyzed using Graphpad Prism (Graphpad) or any other statistical software with two-tailed t-test with Welch’s correction used for statistical analysis. For example, within 24 h of injection into the maternal circulation, CW crossed into the embryonic brain (Figure 5). A CW density of ~400 CW pieces/mm2 corresponds to 4 x 105 CW pieces per entire cortical plate indicating that ~2% of the inoculum into mother trafficked to the fetal brain.